The goal of this project is to analyze various aspects of gene expression in living cells using molecular tools in conjunction with microscopy techniques. A fundamental question in understanding how the mammalian cell nucleus functions and how genes are expressed is what the dynamic properties of nuclear structures and of nuclear proteins are in living cells. We are addressing these issues by generating functional, fluorescently labeled molecules, which can be introduced into cells and visualized in the nucleus of living cells by time-lapse microscopy. Using these methods we have succeeded in visualizing gene expression processes in living cells to study the structure-function relationship of the mammalian cell nucleus. We have demonstrated that transcription and pre-mRNA splicing are spatially tightly linked in vivo. Upon gene activation pre-mRNA splicing factors are recruited from specific intranuclear storage/assembly compartments to transcription sites. This targeting process involves the phosphorylation of pre-mRNA splicing factors most likely by kinases residing in the intranuclear compartments. Recently we have initiated studies to measure the kinetic properties of proteins in vivo by using photobleaching techniques. We have qualitatively and quantitatively analyzed more than 20 nuclear proteins involved in processes such as transcription, pre-mRNA splicing, ribosomal biogenesis, and control of cell proliferation and apoptosis. We find that the vast majority of nuclear proteins move very rapidly within the cell nucleus and that the movement within the nucleoplasm is diffusion mediated. In addition proteins which are enriched in specific nuclear subcompartments, such as the nucleolus, are similarly highly mobile. Resident proteins rapidly and continuously associate and dissociate from nuclear subcompartments, suggesting that the compartments represent the steady-state association/dissociation ratio of the resident proteins. We are using kinetic models and computer simulation to determine the association/dissociation constant, the residence time and the flux of proteins through nuclear compartments. These methods provide powerful tools to now analyze the functional properties of nuclear protein in living cells. We are also using microscopy techniques to study the molecular mechanisms of the pre-mRNA splicing reaction and how RNA processing events are coordinated with transcription. Regarding the latter issue, we have previously shown by time-lapse microscopy that pre-mRNA splicing factors are rapidly targeted to a newly activated transcription site in vivo. Using combined fluorescence in situ hybridization (FISH) and antibody staining techniques we have now shown that splicing factor targeting is dependent on the interaction of splicing factors with the C-terminal domain of the largest subunit of RNA polymerase II. This finding gives further support to the emerging notion that all RNA processing events are spatially complexes with the transcription machinery and has significant implications for the regulation of gene expression. We are using similar FISH/antibody techniques to address the mechanisms of pre-mRNA splicing in vivo. A key feature of the spliceosome is the high degree of intermolecular reorganization of RNA- RNA and RNA-protein interactions required to efficiently carry out the splicing reaction in vivo. It is largely unclear how these reorganizations are regulated in vivo. A candidate for a catalyst of intra-spliceosomal reorganization was recently identified in the form of a spliceosomal cyclophilin protein. In vitro studies to identify a role for this cyclophilin in splicing have been hampered by the low efficiently of in vitro splicing. We noticed in FISH experiments that a specific inhibitor of cyclophilin, cyclosporin A, prevents the release of transcripts from the site of transcription. This block is characteristic of a splicing defect. Using specific splice junction probes we found that the accumulated RNA was indeed unspliced. We have confirmed the reduction in splicing in vivo by RT-PCR. These observations strongly point towards at least one cyclophilin-mediated step in pre-mRNA splicing in vivo. In collaboration with Dr. David Horowitz, Uniformed Services University of the Health Services, Bethesda, MD, we are developing refined in vitro assays to further characterize the role of cyclophilins in pre-mRNA splicing.